Mariana Nunes Barbosa
MULTIMODAL ACTIONS OF BROWN SEAWEED (OCHROPHYTA)
BIOACTIVE COMPOUNDS IN INFLAMMATION AND ALLERGY NETWORK
Thesis for Doctor Degree in Pharmaceutical Sciences
Phytochemistry and Pharmacognosy Specialty
Work performed under the supervision of
Professor Doctor Paula Cristina Branquinho de Andrade
and co-supervision of
Professor Doctor Patrícia Carla Ribeiro Valentão
Study nature, love nature, stay close to nature. It will never fail you. – Frank Lloyd Wright
framework of POPH – QREN – Type 4.1 – Advanced Training, funded by the Fundo Social Europeu (FSE) and by National funds of Ministério da Educação e Ciência (MEC), and by Programa de Cooperación Interreg V-A España–Portugal (POCTEP) 2014–2020 (project 0377_IBERPHENOL_6_E).
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I
T IS AUTHORIZED THE REPRODUCTION OF THIS THESIS ONLY FOR RESEARCH PURPOSES,
UNDER THE WRITTEN STATEMENT OF THE INTERESTED PARTY,
COMMITTING ITSELF TO DO IT.
PUBLICATIONS
The data contained in the following works make part of this thesis.
PUBLICATIONS IN INTERNATIONAL PEER-REVIEWED JOURNALS INDEXED AT THE JOURNAL CITATION REPORTS (JCR) OF THE ISIWEB OF KNOWLEDGE:
1. Barbosa M, Valentão P, Andrade PB. Bioactive compounds from
macroalgae in the new millennium: Implications for neurodegenerative diseases. Mar Drugs. 2014 Sep; 12 (9): 4934–4972.
2. Barbosa M, Collado-González J, Andrade PB, Ferreres F, Valentão P,
Galano JM, Durand T, Gil-Izquierdo Á. Nonenzymatic α-linolenic acid derivatives from the sea: Macroalgae as novel sources of phytoprostanes. J
Agric Food Chem. 2015 Jul ;63 (28): 6466–6474.
3. Barbosa M, Valentão P, Andrade PB. Biologically active oxylipins from
enzymatic and nonenzymatic routes in macroalgae. Mar Drugs. 2016 Jan; 14 (1): 23.
4. Fernandes F, Barbosa M, Oliveira AP, Azevedo IC, Sousa-Pinto I,
Valentão P, Andrade PB. The pigments of kelps (Ochrophyta) as part of the flexible response to highly variable marine environments. J Appl Phycol 2016 Dec; 28 (6): 3689–3696.
5. Barbosa M, Fernandes F, Pereira DM, Azevedo IC, Sousa-Pinto I, Andrade
PB, Valentão P. Fatty acid patterns of the kelps Saccharina latissima,
Saccorhiza polyschides and Laminaria ochroleuca: Influence of changing
environmental conditions. Arab J Chem 2017 (in press). DOI: 10.1016/j.arabjc.2017.01.015.
6. Barbosa M, Lopes G, Ferreres F, Andrade PB, Pereira DM, Gil-Izquierdo
Á, Valentão P. Phlorotannin extracts from Fucales: Marine polyphenols as bioregulators engaged in inflammation-related mediators and enzymes.
Algal Res 2017 Dec; 28: 1–8.
7. Lopes G, Barbosa M, Vallejo F, Gil-Izquierdo Á, Andrade PB, Valentão P,
Pereira DM, Ferreres F. Profiling phlorotannins from Fucus spp. of the Northern Portuguese coastline: Chemical approach by
PUBLICATIONS
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8. Barbosa M, Lopes G, Valentão P, Ferreres F, Gil-Izquierdo Á, Pereira DM,
Andrade PB. Edible seaweeds’ phlorotannins in allergy: a natural multi-target approach. (Under review)
9. Barbosa M, Lopes G, Andrade PB, Valentão P. Inflammation and allergy
network: The multimodal actions of brown seaweed phlorotannins. (Manuscript in preparation)
BOOK CHAPTER:
1. Barbosa M, Valentão P, Andrade PB. Astaxanthin and fucoxanthin:
Promising marine xanthophylls with therapeutic potential. Accepted for
publication in Encyclopedia of Marine Biotechnology, Kim SK (Ed.).
Wiley-Blackwell, New Jersey, USA.
ORAL COMMUNICATION:
1. Barbosa M, Fernandes F, Pereira DM, Valentão P, Ferreres F,
Gil-Izquierdo Á, Andrade PB. UHPLC-QqQ-MS/MS method for phytoprostane profiling in macroalgae. 11th National Meeting of Organic Chemistry and 4th
Meeting of Therapeutic Chemistry. December 1–3, 2015. Porto, Portugal.
POSTER COMMUNICATIONS:
1. Andrade PB, Lopes G, Barbosa M, Weber GM, Pinto E, Valentão P.
Exploring seaweeds: the potential of phlorotannins. 8th ISANH Congress on
Polyphenols Applications. June 5–6, 2014. Lisboa, Portugal.
2. Barbosa M, Collado-González J, Ferreres F, Valentão P, Fernandes F,
Pereira DM, Gil-Izquierdo Á, Andrade PB. Non-enzymatic α-linolenic acid
derivatives in macroalgae: Phytoprostane profiling. 2nd EuCheMS Congress
on Green and Sustainable Chemistry (EuGSC). October 4–7, 2015. Lisboa, Portugal.
3. Andrade PB, Barbosa M, Lopes G, Ferreres F, Gil-Izquierdo Á, Pereira DM, Valentão P. Marine algal polyphenols: Phlorotannin-targeted extracts from Fucus spp. and their anti-inflammatory potential. XXIX International Conference on Polyphenols and 9th Tannin Conference. July 16–20, 2018.
XII AUTHOR STATEMENT
The author declares to have afforded a major contribution to the technical execution, interpretation of the results and manuscript preparation of all works included in this thesis, with the collaboration of other coauthors.
ACKNOWLEDGMENTS
Accomplishing this PhD thesis would not have been possible without the contribution of several people and institutions to whom I would like to thank:
To “Fundação para a Ciência e a Tecnologia” (FCT) for granting me a Doctoral scholarship (SFRH/BD/95861/2013) under the POPH – QREN – Type 4.1 – Advanced Training, funded by the European Social Fund (FSE) and by National funds from the “Ministério da Educação e Ciência”, and by Programa de Cooperación Interreg V-A España–Portugal (POCTEP) 2014–2020 (project 0377_IBERPHENOL_6_E).
To Prof. Doctor Paula Cristina Branquinho de Andrade, my supervisor, for the continuous support of my PhD. I am gratefully indebted to her for accepting me in the Laboratory of Pharmacognosy of the Faculty of Pharmacy of the University of Porto and for all the years of guidance and encouragement. With her, I began my humble path in research, always as her dedicated student. I have always admired her professional journey and her charisma, which incented me to pursue the PhD. Prof. Paula consistently allowed this thesis to be my own work but steered me in the right direction whenever she thought I needed. In fact, without her valuable inputs this thesis could not have been successfully conducted. I could not have imagined having a better supervisor and mentor for my PhD. Thank you.
To Prof. Doctor Patrícia Carla Ribeiro Valentão, co-supervisor of this thesis, for her uninterrupted patience and insightful recommendations. Whenever I ran into a trouble spot or had a question about my research or writing, her door was always open. Prof. Patrícia’s attention to detail drove me to be better and her hard questions incented me to widen my research from various perspectives. For all this, I would like to express my very great appreciation.
To Prof. Doctor Federico Ferreres, from Centro de Edafología y Biología Aplicada del Segura (CEBAS), of Consejo Superior de Investigaciones Científicas (CSIC), Murcia, Spain, for his availability and essential contribution for the identification of phlorotannins by
HPLC-DAD-ESI/MSn and UPLC-ESI-QTOF/MS. His willingness to give his time so
generously has been very much appreciated.
To Prof. Doctor Ángel Gil-Izquierdo, also from CEBAS-CSIC, for his assistance in conducting the UHPLC-QqQ-MS/MS analysis of phytoprostanes.
ACKNOWLEDGMENTS
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To my PhD colleagues and to all the staff of the Laboratory of Pharmacognosy of the Faculty of Pharmacy of the University of Porto, for their companionship, for the stimulating discussions and for contributing to the normal functioning of the lab and the development of this thesis.
To my dear friends that even under the toughest circumstances always made me laugh. I sincerely appreciate your support.
To Lara Reis, my friend of a lifetime, for her precious encouragement and unceasing support.
To my family, particularly my grandmother Belmira, my uncle Serafim, my cousin Ana, my brother, and my nephew Francisco, for their care and support.
To Hugo Santos, for all his dedication, understanding and care. Thank you for always being there.
To my parents, for their never-ending support and unconditional love. None of this would have been possible without them.
ABSTRACT
Among the wealth of biodiversity characterizing the marine environment, macroalgae, commonly addressed as seaweeds, have proved their auspicious ecological roles, as well as their chemical and biological potential. Seaweeds are an abundant and heterogenous group of photosynthetic organisms, distributed worldwide and endowed of unique molecules with high impact in food science, pharmaceutical industry, and public health. Within seaweed groups, the brown ones (Ochrophyta) stand out, as one of the most prolific producers of functional compounds. To harness the biotechnological potential of the Portuguese marine flora, several seaweed species were analyzed and explored for their chemical composition and biological activities. Moreover, as seaweed cultivation has become more widespread, there is a need to expand the knowledge on this material. Therefore, seaweeds grown in integrated multi-trophic aquaculture (IMTA) systems were also studied.
A complex fatty acid profile, characterized mainly by the presence of medium and long fatty acyl chains (14–22 carbon atoms), with different degrees of unsaturation, was observed in Saccharina latissima (Linnaeus) C.E. Lane, C. Mayes, Druehl & G.W.
Saunders, Saccorhiza polyschides (Lightfoot) Batters, and Laminaria ochroleuca Bachelot de la Pylaie tissues subjected to seasonal variations, from different sources (wild and aquaculture), and cultivated at different depths at sea. The specimens of S. latissima, S. polyschides, and L. ochroleuca also exhibited a variable composition in terms of carotenoids and chlorophylls. In these works, a major contribution of surrounding environmental conditions, as well as of species-specific factors, was observed for both fatty acid and pigment composition.
The free phytoprostane composition was assessed, for the first time, in seaweed material through advanced mass spectrometry-based analysis. The profile of phytoprostanes varied greatly, F1t-phytoprostanes and L1-phytoprostanes being the
predominant and the minor classes, respectively. No correlation was observed between the phytoprostane content and the amounts of α-linolenic acid, their known precursor, reinforcing the influence of intra- and inter-species interactions on chemical signatures.
Different species of Fucus, the most prominent and species-rich genus within the order Fucales, were analyzed for their phlorotannin composition. Isomers of
fucophlorethol, dioxinodehydroeckol, difucophlorethol, fucodiphlorethol,
bisfucophlorethol, fucofuroeckol, trifucophlorethol, fucotriphlorethol, tetrafucophlorethol, and of fucotetraphlorethol were tentatively identified in purified extracts of Fucus guiryi Zardi, Nicastro, E.S. Serrão & G.A. Pearson, Fucus serratus Linnaeus, Fucus spiralis
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Linnaeus, and Fucus vesiculosus Linnaeus. The characterized phlorotannins exhibited generally low degree of polymerization (3–6 phloroglucinol units) and belonged mainly to the class of fucophloretols, suggesting a relationship between taxon and the structural type of phlorotannins.
In addition to the chemical profiling, the anti-inflammatory and anti-allergic potential of the phlorotannin-targeted extracts was addressed.
The extracts were able to dose-dependently inhibit lipoxygenase (LOX) activity and to scavenge nitric oxide (NO) radical in cell-free assays, both being correlated with the total phlorotannin content. The overproduction of NO induced by lipopolysaccharide (LPS) in RAW 264.7 macrophages was also efficiently surmounted by non-cytotoxic concentrations of phlorotannin extracts, evidencing their potential benefits in inflammation-related conditions.
Likewise, phlorotannin-targeted extracts from Fucus spp. strongly inhibited allergy-related enzymatic systems. In particular, F. guiryi and F. serratus extracts (the ones with higher phlorotannin amounts) inhibited hyaluronidase (HAase) more efficiently than the reference drug disodium cromoglicate (DSCG). The purified extracts were also able to reduce RBL-2H3 basophils’ degranulation induced by either antibody-antigen complex or by calcium ionophore and, again, strong correlations were found between the above-mentioned effects and the amount of phlorotannins in the extracts.
The outcomes of these studies point to the potential interest of the use of the selected seaweed species as both food and for nutraceutical and/or pharmaceutical applications. In particular, the multi-target capacity addressed to phlorotannin-targeted extracts supports the potential of these polyphenols as valuable naturally occurring pharmacological alternatives with a large spectrum of activity, making high-purity extracts potential candidates for functional foodstuffs.
Keywords: Seaweeds; Fatty acids; Pigments; Phytoprostanes; Phlorotannins;
RESUMO
Entre a incalculável biodiversidade que caracteriza o ambiente marinho, as macroalgas têm provado a sua importância ecológica e o seu promissor potencial biotecnológico. A distribuição destes organismos fotossintéticos é ubiquitária e a heterogeneidade de espécies traduz-se numa vasta diversidade química, com particular relevância na indústria farmacêutica e alimentar. As espécies de macroalgas castanhas (Ochrophyta) destacam-se como fontes prolíficas de compostos funcionais. No sentido de valorizar o potencial biotecnológico da flora algal Portuguesa, diferentes espécies de macroalgas foram exploradas no que respeita à sua composição química e atividades biológicas. Tendo em conta o aumento da produção de macroalgas em aquacultura e a necessidade de aprofundar o conhecimento deste material, foram também objeto de estudo espécies cultivadas em sistemas de aquacultura multi-trófica integrada (IMTA).
Foi observado um perfil de ácidos gordos complexo, caracterizado principalmente
pela presença de ácidos gordos de cadeia média e longa (14–22 átomos de carbono) e com
diferentes graus de insaturação, em tecidos de Saccharina latissima (Linnaeus) C.E. Lane, C. Mayes, Druehl & G.W. Saunders, Saccorhiza polyschides (Lightfoot) Batters, e
Laminaria ochroleuca Bachelot de la Pylaie, desenvolvidos sob a influência de diferentes
fatores. Também ao nível da composição em carotenoides e clorofilas, S. latissima, S.
polyschides e L. ochroleuca apresentaram perfis variáveis. A variabilidade observada no
perfil químico das diferentes algas demonstrou não só a expressão da individualidade de cada espécie, como também a complexa influência de fatores ambientais na distribuição e acumulação de compostos.
Pela primeira vez determinou-se o perfil de fitoprostanos de diferentes espécies de
macroalgas, por espetrometria de massa. Nas amostras analisadas, F1t-fitoprostanos e L1
-fitoprostanos foram, respetivamente, os compostos maioritários e minoritários. Não se observou qualquer correlação entre os níveis de fitoprostanos nas amostras e o conteúdo do seu percursor (ácido α-linolénico), reforçando, uma vez mais, a relevância de fatores intra e interespecíficos para a composição química.
Diferentes espécies de algas do género Fucus, o mais proeminente e rico em espécies dentro da ordem Fucales, foram analisadas relativamente à sua composição em florotaninos. Isómeros de fucofloretol, dioxinodesidroecol, difucofloretol, fucodifloretol, bisfucofloretol, fucofuroecol, trifucofloretol, fucotrifloretol, tetrafucofloretol, e de fucotetrafloretol foram tentativamente identificados nos extratos purificados de Fucus
guiryi Zardi, Nicastro, E.S. Serrão & G.A. Pearson, Fucus serratus Linnaeus, Fucus spiralis Linnaeus e Fucus vesiculosus Linnaeus. Os florotaninos aqui caracterizados
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apresentaram, em geral, um baixo grau de polimerização (3–6 unidades de floroglucinol), sendo a maioria pertencente à classe dos fucofloretóis, sugerindo a existência de uma relação quimiotaxonómica.
Para além do estudo da composição química, foi também explorado o potencial anti-inflamatório e anti-alérgico dos extratos purificados de florotaninos.
Em sistemas não celulares, os extratos inibiram a atividade da enzima lipoxigenase (LOX) e sequestraram o radical óxido nítrico (NO), de forma dependente da concentração. Ambos os efeitos mostraram estar correlacionados com o teor de florotaninos dos extratos. Concentrações não citotóxicas dos extratos foram capazes de reduzir o NO produzido pela linha celular de macrófagos RAW 264.7, quando estimulados com lipopolissacarídeo bacteriano (LPS), evidenciando os seus potenciais benefícios em patologias com um processo inflamatório associado.
De igual modo, os extratos purificados de florotaninos inibiram enzimas relevantes em condições alérgicas. Os extratos obtidos de F. guiryi e F. serratus, aqueles com maior conteúdo de florotaninos, foram particularmente promissores e mais eficientes na inibição da hialuronidase (HAase) do que o fármaco de referência, cromoglicato dissódico (DSCG). Os extratos purificados reduziram também a desgranulação de basófilos RBL-2H3 quando estimulados por um complexo anticorpo-antigénio ou por um ionóforo de cálcio e, uma vez mais, foi observada a existência de forte correlação entre os efeitos descritos e o teor de florotaninos nos extratos.
Os principais resultados destes estudos apontam para o potencial promissor do uso das espécies de algas selecionadas, quer como alimento, quer para aplicações nutracêuticas e/ou farmacêuticas. Em particular, a capacidade de atuação em múltiplos alvos dos extratos purificados de florotaninos reforça o potencial destes polifenóis como alternativas farmacológicas de origem natural com um grande espetro de atividade, tornando extratos de elevado grau de pureza candidatos interessantes para o desenvolvimento de formulações farmacêuticas e/ou de alimentos funcionais.
Palavras-chave: Macroalgas; Ácidos gordos; Pigmentos; Fitoprostanos; Florotaninos;
TABLE OF CONTENTS
PUBLICATIONS ... IX ACKNOWLEDGMENTS ... XIII ABSTRACT ... XVII RESUMO ... XXI LIST OF FIGURES ... XXXI LIST OF TABLES ... XXXVII LIST OF ABBREVIATIONS ... XLI THESIS OUTLINE ... 1 CHAPTER I–INTRODUCTION AND OBJECTIVES
1.Introduction ... 5 1.1. The marine biome: A treasure of biological and chemical diversity ... 5 1.1.1. Bioprospecting seaweeds for human health: Challenges and opportunities ... 6 1.1.2. Seaweeds and the Portuguese seascape ... 8 1.2. Brown seaweeds: A special focus ... 10 1.2.1. The order Laminariales ... 10 1.2.2. The order Fucales ... 12 1.2.3. Functional compounds ... 14 1.2.3.1. Fatty acids and oxidation derivatives ...15 1.2.3.1.1. Non-enzymatically-derived algal oxylipins: The phytoprostanes ... 18 1.2.3.1.2. Extraction and profiling of fatty acids and phytoprostanes ... 21 1.2.3.2. Pigments: Chlorophylls and carotenoids ... 22 1.2.3.2.1. Pigment extraction and profiling ... 27 1.2.3.3. Phlorotannins ... 28 1.2.3.3.1. Extraction, purification, and profiling ... 32 1.3. General overview into inflammation and allergy network ... 35 1.3.1. Inflammation and multimodal actions of phlorotannins on inflammatory responses ... 36 1.3.2. Allergy and modulation of allergic events by phlorotannins ... 46 2.Objectives ... 53 CHAPTER II–EXPERIMENTAL SECTION
3.Experimental Section ... 57 3.1. Standards and reagents ... 57 3.2. Sampling... 59
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3.3. Fatty acids and pigments ...60 3.3.1. Algal material ...60 3.3.2. Fatty acid extraction and derivatization ... 62 3.3.3. GC/IT-MS qualitative analysis of fatty acids ... 62 3.3.4. Fatty acid quantification by GC-FID ... 63 3.3.5. Pigment extraction ... 64 3.3.6. HPLC-DAD analysis of pigments ... 65 3.4. Phytoprostanes ... 65 3.4.1. Algal material ... 65 3.4.2. Phytoprostane extraction ... 66 3.4.3. UHPLC-QqQ-MS/MS analysis of free phytoprostanes ... 68 3.5. Phlorotannin purified extracts: Composition and biological activity ... 68 3.5.1. Preparation of phlorotannin purified extracts ... 69 3.5.2. Phlorotannin quantification ... 70 3.5.3. Phlorotannin qualitative profiling ... 71 3.5.3.1. HPLC-DAD-ESI/MSn analysis... 71 3.5.3.2. UPLC-ESI-QTOF/MS analysis ... 71 3.5.4. Biological effects of phlorotannin purified extracts ... 72 3.5.4.1. Anti-inflammatory activity ... 72 3.5.4.1.1. Cell assays ... 72 3.5.4.1.1.1. Cell culture conditions and treatments ... 72 3.5.4.1.1.2. Cell viability ... 72 3.5.4.1.1.3. Nitric oxide determination ... 73 3.5.4.1.2. Cell-free assays ... 74 3.5.4.1.2.1. Nitric oxide radical scavenging capacity ... 74 3.5.4.1.2.2. Lipoxygenase inhibition ... 74 3.5.4.2. Anti-allergic activity ... 75 3.5.4.2.1. Cell assays ... 75 3.5.4.2.1.1. Cell culture conditions and assays ... 75 3.5.4.2.1.2. A23187-mediated cell degranulation ... 76 3.5.4.2.1.3. IgE/antigen-mediated cell degranulation ... 76 3.5.4.2.1.4. MTT reduction assay ... 76 3.5.4.2.1.5. Crystal violet staining assay ... 76 3.5.4.2.1.6. Determination of β-hexosaminidase released ... 77 3.5.4.2.1.7. Determination of histamine released ... 77 3.5.4.2.2. Cell-free assays ... 78 3.5.4.2.2.1. β-Hexosaminidase inhibition ... 78 3.5.4.2.2.2. Hyaluronidase inhibition... 78
CHAPTER III–RESULTS AND DISCUSSION
4.Results and Discussion ... 83 4.1. Influence of changing environmental conditions on fatty acid and pigment composition of the kelps L. ochroleuca, S. latissima, and S. polyschides ... 83 4.1.1. Fatty acids ... 83 4.1.1.1. General overview ... 83 4.1.1.2. Seasonal variation vs fatty acid profile ... 94 4.1.1.3. Wild natural stocks vs IMTA ... 95 4.1.1.4. Cultivation depth vs fatty acid profile ... 98 4.1.2. Pigments ... 100 4.1.2.1. General overview ... 100 4.1.2.2. Harvesting period and origin vs pigment profile ... 104 4.1.2.3. Pigment distribution within thalli ... 105 4.1.2.4. Pigment composition of S. latissima tissues vs cultivation depth ... 105 4.2. Non-enzymatic α‑linolenic acid derivatives from the sea: Macroalgae as novel sources of phytoprostanes ... 107 4.2.1. Occurrence of α‑linolenic acid in macroalgae ... 107 4.2.2. Occurrence of free phytoprostanes in macroalgae ... 107 4.2.3. Free phytoprostanes vs α‑linolenic acid ... 113 4.3. Phlorotannins ... 114 4.3.1. Phlorotannin profile of Fucus spp. ... 114 4.3.1.1. Phlorotannin trimers ... 120 4.3.1.2. Phlorotannin tetramers ... 122 4.3.1.3. Phlorotannin pentamers ... 123 4.3.1.4. Phlorotannin hexamers ... 124 4.3.2. Phlorotannin extracts from Fucales as bioregulators engaged in inflammation-related mediators and enzymes. ... 125
4.3.2.1. Quantitative overview ... 125 4.3.2.2. Anti-inflammatory activity ... 127 4.3.2.2.1. Lipoxygenase inhibitory potential ... 127 4.3.2.2.2. Effect on inflammatory mediators ... 129 4.3.3. Phlorotannin extracts from Fucales in allergy network. ... 134 4.3.3.1. Effect of phlorotannin purified extracts on cell degranulation ... 134 4.3.3.1.1. A23187-mediated cell degranulation ... 138 4.3.3.1.2. IgE/antigen-mediated cell degranulation ... 139 4.3.3.2. Allergy-related enzymes ... 143 4.3.3.2.1. β-Hexosaminidase inhibition ... 143 4.3.3.2.2. Hyaluronidase inhibition ... 145
TABLE OF CONTENTS XXVIII CHAPTER IV–CONCLUSIONS 5.Conclusions ... 149 CHAPTER V–REFERENCES 6.References ... 153
LIST OF FIGURES
Figure 1. Schematic representation of an IMTA system ... 8 Figure 2. Laminaria ochroleuca (A), Saccharina latissima (B), and a young specimen of Saccorhiza polyschides attached to rocks (C) ... 12 Figure 3. Fucus vesiculosus (A) and its air-filled bladders (B) ... 13 Figure 4. Chemical structures of the main polyunsaturated fatty acids found in
macroalgae ... 16
Figure 5. Non-enzymatic formation of phytoprostanes from α-linolenic acid ... 20 Figure 6. Conversion of chlorophyll a to pheophytin a catalyzed by Mg-dechelatase
under acidic conditions... 23
Figure 7. Chemical structure of the main c-type chlorophylls in seaweeds ... 24 Figure 8. Hypothetical fucoxanthin biosynthetic pathway in brown macroalgae ... 26 Figure 9. Proposed biosynthetic pathway of phloroglucinol and oxidative coupling
between pairs of phloroglucinol free radical units to form the first dimeric phlorotannins……….……….. 29
Figure 10. Structures of representatives of each phlorotannin class, highlighting their
distinctive chemical features ... 31
Figure 11. 2,4-Dimethoxybenzaldehyde (DMBA) colorimetric reaction ... 34 Figure 12. Schematic representation of the main allergy and inflammation targets of
phlorotannins ... 39
Figure 13. Studied Portuguese waters with the sampling sites marked with numbers .... 59 Figure 14. Schematic representation of the protocol for phytoprostane extraction from
macroalgae samples and their profiling by UHPLC-QqQ-MS/MS ... 67
Figure 15. Schematic representation of the general procedure for obtaining phlorotannin
purified extracts from Fucus spp. and their profiling by HPLC-DAD-ESI/MSn and
UPLC-ESI-QTOF/MS ... 69
Figure 16. Schematic representation of the general procedure for obtaining phlorotannin
purified extracts from Fucus spp. for biological assessment... 70
Figure 17. Reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) to formazan, by mitochondrial dehydrogenases of metabolically active cells ... 73
Figure 18. Nitrite (NO2-) determination by Griess assay ... 74
Figure 19. Linoleic acid oxidation to 13-hydroperoxy-linoleic acid catalyzed by
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Figure 20. β-Hexosaminidase-catalyzed conversion of p-nitrophenyl
N-acetyl-β-D-glucosaminide into N-acetyl-β-D-N-acetyl-β-D-glucosaminide and the yellow p-nitrophenolate product………77
Figure 21. Morgan-Elson reaction applied to the determination of hyaluronidase (HAase)
activity ... 79
Figure 22. Representative GC/IT-MS chromatogram of the fatty acid profile of
whole-specimen samples of L. ochroleuca (Lo_W_Am_Apr13). ... 84
Figure 23. Sum of saturated (ΣSFA), monounsaturated (ΣMUFA), polyunsaturated
(ΣPUFA) and total fatty acid content (ΣFA) in L. ochroleuca, S. latissima and S.
polyschides chloroform:methanol extracts (mg/kg dry algae) ... 85 Figure 24. Projection of S. latissima (A1), S. polyschides (B1) and L. ochroleuca (C1),
under the influence of different parameters and loadings (A2, B2 and C2) by fatty acid composition into the plane composed by the principal components PC1 and PC2 containing 63.0, 100.0 and 69.7% of the total variance for S. latissima, S. polyschides and
L. ochroleuca, respectively... 93 Figure 25. n-3 and n-6 PUFA content (mg/kg dry algae), and n-6/n-3 ratio of L. ochroleuca, S. latissima and S. polyschides chloroform:methanol extracts ... 97 Figure 26. Sum of saturated (ΣSFA), monounsaturated (ΣMUFA), polyunsaturated
(ΣPUFA) and total fatty acid content (ΣFA) of S. latissima tissues cultivated at 5, 10, and 15 m deep subjected to different light intensity and sea surface temperatures (SST) ... 99
Figure 27. HPLC-DAD carotenoid and chlorophyll profiles of acetone extracts from L. ochroleuca (Lo_W_IMTA_Jan13), S. latissima (Sl_W_IMTA_Apr13) and S. polyschides
(Sp_W_N_Jan13) ... 101
Figure 28. Representative UHPLC-QqQ-MS/MS chromatogram of detected
phytoprostanes (C. tomentosum) (A), presumed fragmentation and MRM transitions for
quantification of 9-F1t-phytoprostane (B), 9-epi-9-F1t-phytoprostane (C), 16-B1
-phytoprostane (D), and 9-L1-phytoprostane (E) ... 111
Figure 29. Projection of macroalgae (A)and loadings (B) by phytoprostane composition into the plane composed by principal components PC1 and PC2 containing 92.9% of the total variance ... 112
Figure 30. Extracted Ion Chromatograms (EIC) obtained from HPLC-DAD-ESI/MSn of purified phlorotannin extracts from wild-sourced and aquaculture-grown F. vesiculosus (Fves-w and Fves-a, respectively), F. guiryi (Fg), F. serratus (Fser), and F. spiralis (Fspi)………117
Figure 31. Proposed fragmentation patterns of the structures tentatively identified in
phlorotannin purified extracts from Fucus spp. ... 121
Figure 32. Mass spectra analysis of the pentamer 18 detected in wild-sourced F. vesiculosus (Fves-w). ... 124 Figure 33. Phlorotannin content in the purified extracts from Fucus spp. ... 126
Figure 34. Lipoxygenase (LOX) inhibition of phlorotannin purified extracts in cell-free
assay ... 127
Figure 35. Effect of phlorotannin purified extracts on the viability and NO levels of RAW
264.7 cells pre-treated for 2 h with the extracts, followed by 22 h co-treatment with LPS (1 μg/mL) or vehicle (culture medium) ... 130
Figure 36. Nitric oxide radical (•NO) scavenging of phlorotannin purified extracts in
cell-free assay ... 133
Figure 37. Effect of IgE/antigen (A) and calcium ionophore A23187 (B) on the cell
viability (MTT reduction), and on β-hexosaminidase and histamine released from RBL-2H3 cells ... 136
Figure 38. Effect of phlorotannin purified extracts on the viability of RBL-2H3 cells with
and without stimulation by the calcium ionophore A23187 or by IgE/antigen ... 137
Figure 39. Effect of phlorotannin purified extracts on β-hexosaminidase and histamine
released from RBL-2H3 cells when stimulated with the calcium ionophore A23187 ... 138
Figure 40. Effect of phlorotannin purified extracts on β-hexosaminidase and histamine
released from RBL-2H3 cells when stimulated with IgE/antigen ... 140
Figure 41. β-Hexosaminidase inhibition of phlorotannin purified extracts in cell-free
systems ... 144
Figure 42. Hyaluronidase (HAase) inhibition of phlorotannin purified extracts in
LIST OF TABLES
Table 1. Anti-inflammatory effects of phloroglucinol (basic unit) and phlorotannins
isolated from brown seaweeds ... 40
Table 2. Anti-inflammatory effects of phlorotannin-rich extracts/fractions obtained from
brown seaweeds ... 43
Table 3. Anti-allergic effects of phloroglucinol (basic unit) and phlorotannins isolated
from brown seaweeds ... 49
Table 4. Anti-allergic effects of phlorotannin-rich extracts/fractions obtained from brown
seaweeds ...51
Table 5. Characterization of macroalgae material used for fatty acid and pigment
analysis……….. 61
Table 6. Regression equations, r2, linearity, limit of detection (LOD), and limit of
quantification (LOQ) for FAME with the employed analytical conditions ... 64
Table 7. Characterization of macroalgae samples used for phytoprostane analysis ... 66 Table 8. Characterization of macroalgae samples used for phlorotannins analysis and
biological studies ... 69
Table 9. Saturated fatty acid content of L. ochroleuca, S. latissima and S. polyschides
chloroform:methanol extracts (mg/Kg dry algae) ... 88
Table 10. Unsaturated fatty acid content of L. ochroleuca, S. latissima and S. polyschides
chloroform:methanol extracts (mg/Kg dry algae) ... 90
Table 11. Carotenoids and chlorophylls content in L. ochroleuca, S. latissima and S. polyschides extracts (mg/kg dry algae) ... 102 Table 12. α-Linolenic acid (g/Kg dry algae) and phytoprostane (ng/100 g dry algae)
content in the analyzed macroalgae species ... 109
Table 13. Retention times (Rt), molecular formula and mass spectrometric data of
molecular ions and main observed fragments of phlorotannins in the extracts of wild-sourced F. vesiculosus (1, 7, 14–19, 21, 22), aquaculture-grown F. vesiculosus (5, 7, 13,
20, 22), F. guiryi (4–6, 8, 10–12), F. serratus (2, 5, 9, 11) and F. spiralis (2, 3, 7, 10)……….……. 118 Table 14. IC50 values found for the phlorotannin purified extracts on LOX inhibition .. 129
Table 15. IC50 values found for the phlorotannin purified extracts on NO levels in RAW
264.7 cell culture medium ... 131
Table 16. IC50 values found for the phlorotannin purified extracts on •NO
scavenging………. 134
Table 17. IC50 values found for the phlorotannin purified extracts on β-hexosaminidase
LIST OF TABLES
XXXVIII
Table 18. IC50 values found for the phlorotannin purified extracts on β-hexosaminidase
and histamine released by IgE/antigen-challenged RBL-2H3 cells ... 139
Table 19. IC50 values found for the phlorotannin purified extracts on β-hexosaminidase
inhibition ... 144
Table 20. IC50 values found for the of phlorotannin purified extracts on hyaluronidase
LIST OF ABBREVIATIONS
β-Hex β-Hexosaminidase
AA Arachidonic acid
AD Alzheimer’s disease
ALA δ-Aminolevulinic acid
AP-1 Activator protein-1
APC Antigen presenting cells
BHA Butylated hydroxyanisole
BHT Butylated hydroxytoluene
BIS-TRIS Bis(2-hydroxyethyl)-amino-tris-(hydroxymethyl)-methane
BMCMC Bone marrow-derived cultured mast cells
BSA Bovine serum albumin
CD23 Low-affinity IgE receptor
CNS Central nervous system
COX Ciclooxygenase
CRA-1 Anti-human FcεRI antibody
CVS Crystal violet staining
DAD Diode array detection
DE Dry extract
DMAB 4-Dimethylaminobenzaldehyde
DMBA 2,4-Dimethoxybenzaldehyde
DMEM Dulbecco's Modified Eagle Medium
DMSO Dimethyl sulfoxide
DNP Dinitrophenyl
DOXP/MEP 1-Deoxyxylulose 5-phosphate/2-C-methylerithrytol 4-phosphate
DSCG Disodium cromoglicate
EBSS Earle’s Balanced Salt Solution
EI Electron impact
EIC Extracted Ion Chromatogram
ERK Extracellular signal-regulated kinase
ESI Electrospray ionization
FAME Fatty acid methyl esters
FBS Fetal bovine serum
FcεRI High-affinity IgE receptor
GC/IT-MS Gas chromatography/ion trap-mass spectrometry
LIST OF ABBREVIATIONS
XLII
GGPP Geranyl geranyl pyrophospate
HA Hyaluronic acid
HAase Hyaluronidase
HBSS Hanks' Balanced Salt Solution
HFD High-fat diet
HMBG High mobility group box protein
HPLC High performance liquid chromatography
HUVEC Human umbilical vein endothelial cells
ICAM Intercellular adhesion molecule
ICR Institute of Cancer Research
IgE Immunoglobulin E
IL Interleukin
IMTA Integrated multi-trophic aquaculture
iNOS Inducible nitric oxide synthase
IκB-α Inhibitor κB-α
JNK c-Jun N-terminal kinase
LC Liquid chromatography
LLE Liquid-liquid extraction
LLP Liquid-liquid partition
LOD Limit of detection
LOQ Limit of quantification
LOX Lipoxygenase
LPS Lipopolysaccharide
LTB4 Leukotriene B4
MAPK Mitogen-activated protein kinases
MCP-1 Monocyte chemoattractant protein-1
MDA Malondialdehyde
MMP Matrix metalloproteinase
MRM Multiple reaction monitoring
MS Mass spectrometry
MTBE Methyl tert-butyl ether
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MUFA Monounsaturated fatty acids
MVA Mevalonate
NF-κB Nuclear factor-κB
NP Normal phase
OPA o-Phthalaldehyde
OXA Oxazolone
PAR Photosynthetically active radiation
PC Phosphatidylcholine
PCA Principal component analysis
PG Prostaglandin
PGE Phloroglucinol equivalents
PI3K/Akt Phosphatidylinositol 3-kinase/protein kinase B
PKS Polyketide synthase
PLA2 Phospholipase A2
PMA Phorbol 12-myristate 13-acetate
PPAR Peroxisome proliferator-activated receptor
PS Photosystem
PTFE Polytetrafluoroethylene
PUFA Polyunsaturated fatty acids
QTOF Quadrupole time-of-flight
rhIL-1α Recombinant human interleukin-1α
ROS Reactive oxygen species
RP Reverse phase
SFA Saturated fatty acids
SLE Solid-liquid extraction
SNP Sodium nitroprusside dehydrate
SPE Solid-phase extraction
SST Sea surface temperature
TQD Tandem quadrupole detector
Th T helper
THF Tetrahydrofuran
TLR Toll-like receptor
TNF Tumor necrosis factor
TPA 12-O-tetradecanoyl-phorbol-13-acetate
UAE Ultrasound-assisted extraction
UCP-1 Uncoupling protein-1
UFA Unsaturated fatty acids
UHPLC-QqQ-MS/MS
Ultra-high performance liquid chromatography coupled to triple-quadrupole mass spectrometry
UV-vis Ultraviolet-visible
LIST OF ABBREVIATIONS
XLIV
WHO World Health Organization
THESIS OUTLINE
This thesis is composed of five main sections:
CHAPTER I–INTRODUCTION AND OBJECTIVES
This chapter provides a broad and state-of-the art overview of the main topics from the workplan of this thesis, including information required to understand the experimental findings. The first part of the Introduction addresses the raising global interest in the use of marine functional ingredients, with special focus on those from brown seaweeds (Ochrohpyta), notwithstanding the main challenges that hinder the full exploitation of these bioresources of the oceans. Furthermore, the occurrence, biosynthesis, chemistry, and biotechnological challenges of some of the most promising functional compounds of Ochrophyta are explored in this section. The last part of this chapter encompasses a general description of the inflammatory and allergic processes, and a review of the literature concerning phlorotannins and their capacity to act upon different critical steps of both inflammatory and allergic response.
The main objectives of this thesis are listed at the end of Chapter I.
CHAPTER II–EXPERIMENTAL SECTION
This section contains information on sampling procedures and experimental protocols employed for the assessment of the parameters under study.
CHAPTER III–RESULTS AND DISCUSSION
This chapter displays the main outcomes of the different works, and the approached subjects are discussed in light of the current knowledge.
CHAPTER IV–CONCLUSIONS
The main conclusions drawn from this thesis are summarized in this chapter.
CHAPTER V–REFERENCES
CHAPTER I
I
NTRODUCTIONO
BJECTIVES1. Introduction
1.1. The marine biome: A treasure of biological and chemical diversity
Oceans dominate the earth’s surface (71%) and harbor a wide variety of living organisms, rendering them prolific reservoirs of chemical diversity. Marine organisms thrive in a complex seawater environment, characterized by broad fluctuations of light (from complete darkness to extensive photic zones), and pressure (1 to over 1 000 atmospheres), also facing huge ranges of temperature (from -1.5 °C in ice sea to 350 °C in deep hydrothermal systems), and nutrients, in cohabitation with a high number of different species (1).
The ability to endure and adapt to a subset of surrounding factors (biotic and abiotic) relies on different adaptation mechanisms of organisms, such as the production of biologically active secondary metabolites. Unlike primary metabolism, that furnishes intermediates for the synthesis of macromolecules directly involved in growth and development of an organism, secondary metabolism is of restricted distribution and an expression of the individuality of the species (2). Secondary metabolites fulfill pivotal functions, maintaining an intricate balance with the multivariate ecological changes and mediating interactions with other organisms. Nevertheless, evidence suggests that many compounds, once considered to be strict secondary metabolites, are now known to act in both primary and secondary roles (e.g., brown seaweed phlorotannins) (3). The chemical diversity of bioactive compounds mirrors the genetic diversity of organisms and the complex habitat in which they are discovered (4). It can then be argued that the chemical profile of an organism, population or community represents an alternative source of information, providing a broad perspective of how environmental changes or pressures may influence the synthesis and activity of primary and secondary metabolites (5). Notwithstanding the eco-physiological roles, what makes marine natural products of great relevance for humans is the uniqueness of their molecular structures and the potency of their biological effects, providing important chemical scaffolds for the discovery of new drugs for the management of numerous diseases.
The marine ecosystem has then raised great curiosity and interest in several scientific fields and industries, as a prospective source for new potential drug leads. Nevertheless, research on the pharmacological properties of marine organisms is limited, and most of it remains unexplored (6). The first serious effort on studying marine natural products dates from 1951 with the pioneering work of Bergman and Feeney (7) that resulted on the isolation of two nucleoside derivatives (spongothymidine and spongouridine) from the
INTRODUCTION
6
sponge Tectitethya crypta de Laubenfels (formely known as Cryptotethya crypta de Laubenfels). This finding led to the synthesis of cytarabine or Ara-C, a spongothymidine analogue clinically approved in 1969 and still currently used to treat different types of leukemia (8). In fact, marine natural products have their stronghold in anticancer chemotherapy: of the seven marine pharmaceuticals in current clinical use, four are anticancer drugs (8). However, the prospects of yielding novel marine-derived compounds with other valuable clinical applications are promising, as the number of compounds isolated from marine organisms reaches now almost 30 000, with hundreds of new ones being discovered every year (9).
1.1.1. Bioprospecting seaweeds for human health: Challenges and opportunities
Although it is true that bioprospecting of marine sources has provided some structurally unique marine products, the search for new biologically active compounds can be considered an almost unlimited field. The increasingly consumer awareness and demand for bio-based products has also redirected efforts towards marine bioprospecting activities and, alongside the pharmaceutical applications, both nutraceutical and cosmeceutical industries have been devoting many resources into the incorporation of marine natural products as functional ingredients (8,10). Contrary to the high-risk/high-reward pharmaceutical market, nutraceuticals and cosmeceuticals have, in general, a rapid route to commercialization, offering low risk and quicker potential return on investment (8). In fact, the global cosmetic market is now estimated to reach 675 billion dollars by 2020 (www.researchandmarkets.com/research/f2lvdg/global_cosmetics), and the global nutraceutical market, comprised of functional foods and beverages and dietary
supplements, should reach 285 billion dollars by 2021
(www.researchandmarkets.com/research/8ltg7l/nutraceuticals), with marine-based
products progressively occupying a large market share.
Interestingly, this renewed focus on marine bioresources relies on the ancient knowledge and empirical use of indigenous communities that have been dependent on these resources for food, medicine, and livelihood (11,12). Examples of early utilization of seaweeds (also addressed as marine algae or macroalgae) for medicinal purposes comprise the Chinese use of some species to treat thyroid-related diseases, such as goiter (16th
century, Chinese herbal, ‘Pen Tsae Kan Mu’), Gelidium spp. for intestinal afflictions and dehydrated Laminaria spp. stipes for the dilation of the cervix in difficult childbirths (13). Seaweeds, in particular, have been an important part of the human diet all around the globe: in Pacific and Asian cultures, seaweeds have long been consumed in a variety of
dishes; in Europe, the traditional consumption of seaweed-based foods was limited to some few countries, like Iceland, Wales and France, but recent trends have shown an increasing acceptance of seaweeds in the Western diet (10,14). Although still largely dominated by Asian countries, seaweed exploitation accounts now for a billionaire market (15). In 2014, about 28.5 million tons of seaweeds and microalgae were harvested for various purposes, including direct consumption or further processing for food, as well as for use as fertilizers and in pharmaceuticals and cosmetics (15). Over the years, promising insights into the bioactivities of extracts, fractions, and isolated compounds from seaweeds have been detailed in numerous reviews (16–25), opening doors for the development of seaweed-derived products with commercial potential.
Despite this global growing interest in the use of marine functional ingredients, there are still many challenges ahead that must be overcome for the full exploitation of marine resources. In general, the supply problem has hampered the research of marine bioactive compounds, mainly because secondary metabolites occur at relatively low concentrations, and their production varies in the same species and even within different parts of a single specimen, as result of variable environmental conditions (26,27). As means of ensuring and improving the supply of these high-value chemicals, different biotechnological approaches have been developed and optimized (28,29). Large-scale cultivation of seaweeds has been a tremendous case of success, having nearly tripled between 2000 and 2014, from 9.3 to almost 27 million tons, and providing more than 95% of the harvested seaweeds around the globe (15). Seaweeds can grow with little or no demand on fresh water in production cycles, and their growth rates exceed those of terrestrial plants (30). However, intensive aquaculture practices of single-species farms have raised some concerns, especially regarding the discharge of nutrients to coastal areas, potentially responsible for deterioration of local marine communities (31,32).
The use of ecological engineering tools, such as integrated multi-trophic aquaculture (IMTA) systems, has arisen as a practical approach for mitigating the environmental impact of monocultures (33–35), but there is still a need for regulation and establishment of ‘‘good practices” for seaweed harvesting, management, and cultivation to enhance the sustainability in the use of ecological goods and services that coastal zones provide (36). IMTA is a flexible system that aims to replicate a small-scale ecosystem, where species from different trophic or nutritional levels are incorporated, and that can be executed in both open water and land-based systems (37). In IMTA systems, seaweeds function as
extractive components within a cultivation food web, assimilating fish-excreted ammonia (NH3), phosphate (PO43-) and carbon dioxide (CO2), and converting them into potentially
INTRODUCTION
8
is possible to manipulate key factors (e.g., light intensity and nutrient loading), allowing a high control over biomass traceability, quality, and security of supply, which are major requisites of the emergent market of functional products from seaweeds for human use (39,40).
Figure 1. Schematic representation of an IMTA system.
1.1.2. Seaweeds and the Portuguese seascape
Seaweeds are abundant and potentially renewable bioresources of the oceans. They
drive the biodiversity and functioning of many shallow benthic ecosystems, accounting for
up to 10% of the global oceanic primary production (41). Seaweeds comprise a heterogenous group of photosynthetic eukaryotic organisms, with more than 10 000 species worldwide (42). There are three macroalgae phyla, conventionally established according to their morphological pigmentation: Rhodophyta (red seaweeds), Chlorophyta (green seaweeds), and Ochrophyta (brown seaweeds). The color of Chlorophyta is due to the presence of chlorophylls a and b in the same proportions as in terrestrial higher plants. The greenish brown color of Ochrophyta is essentially attributed to the presence of fucoxanthin, combined with chlorophylls a and c. Phycobilins, such as phycoerythrin and phycocyanin, are responsible for the color of Rhodophyta (16). Besides their thallus color, seaweeds differ considerably in many ultrastructural components (e.g., presence/absence, number, and position of flagella) and biochemical features (e.g., composition of cell walls, and storage compounds) (4).
Unlike green algae, commonly found in freshwater, and even in terrestrial locations, red and brown algae are both almost exclusively marine (43). Global distribution of seaweed species is highly dynamic and physiologically constrained by nutrient availability, temperature, and light (44). Moreover, climate-related stressors, intensive fishing and
Water discharge NH3 PO4 3-CO2 Seaweed pond
Feed Clean water
NH3 PO4 3-CO2 Solid wastes Fish pond O2 O2 Light Mechanical filter
other anthropogenic activities have deeply impacted nearshore ecosystems for centuries, leading to significant changes in the structure and functioning of seaweed natural beds (45).
The coast of mainland Portugal is one of the longest in the European Union, with approximately 830 km long, and a hotspot of marine diversity (46). The Portuguese coastline is subjected to particular biogeographic circumstances, receiving climatic influences from both the Atlantic Ocean and the Mediterranean Sea, which determine unique combinations of species forming macroalgal communities (46,47). In fact, a marked gradient in the distribution of macroalgal flora is evidenced: the flora of the Northern plateau is similar to that found in Central Europe (Brittany and South of the British Isles), whereas in the south, the algal flora is quite different, with a clear influence of the Mediterranean and of the North zone of the West African coast (48). Despite its biogeographic importance, the macroalgal flora of this region has not been thoroughly studied; the latest updated checklist of the benthic marine macroalgal dates back to 2009, reporting the presence of 320 species (200 Rhodophyta, 70 Ochrophyta, and 50 Chlorophyta) in the Northern Portuguese coast (47).
In Portugal, the major economic activities related to the sea are shipbuilding,
shipping, and fishing; however, and, despite the high diversity of species, macroalgae are still one of the least studied and exploited aquatic resources (49). Back in the seventies, Portugal was one of the leading producers of agar (a hydrophilic colloid extracted from certain seaweeds of Rhodophyta) in the world, but the situation has changed considerably, Indonesia and China emerging as the largest producers of agar-bearing seaweeds and agar manufacturers, achieved by means of in- and offshore cultivation (50). The country’s recent focus on aquaculture development opened doors for macroalgal cultivation (38,51), and the need to expand the knowledge on this material. Recently, some young companies, together with research groups from Academia, have been promoting numerous initiatives to harness the biotechnological potential of the Portuguese marine flora (52), and considerable efforts were made on the search of functional compounds from native macroalgae species during the last decade (53–68).
Among macroalgae, Rhodophyta and Ochrophyta are known to be the most prolific producers of secondary metabolites (9,69). The latest annual “Marine Natural Products” review (9) reported the discovery of 33 new compounds from both red and brown algae, highlighting the current interest in Ochrophyta phylum, for which a large number of studies concerning biological activities is available.
INTRODUCTION
10 1.2. Brown seaweeds: A special focus
Ochrophyta represents the second largest group of seaweeds, comprising approximately 1 800 species (42). Both red and green algae originated from a primary endosymbiosis between a prokaryotic photosynthetic cyanobacterium and a non-photosynthetic eukaryotic protist host, whilst brown algae developed from a secondary endosymbiotic process involving a non-photosynthetic eukaryote and a unicellular red alga (70). Therefore, brown algae (kingdom Chromista) belong to a lineage phylogenetically distant from the red and green algae (kingdom Plantae), holding quite distinctive characteristics, such as wall composition (alginates and sulfated fucans) (71), carbon storage compounds (laminarin and mannitol) (72), the ability to synthetize both plant-like (C18) and animal-like (C20) oxylipins (73), as well as a number of lateral gene
transfer events that have shaped their metabolism (74,75).
In general, brown seaweeds exhibit pronounced spatiotemporal variability and a wide range of sizes, including the largest of all algae. The large size of much brown seaweed and their distribution along the rocky intertidal and subtidal zones have made them very suitable subjects for human study, i.e. the access to most components is easily accomplished, and the large size allows the extraction, in large amounts, of the associated bioactive compounds (4).
Among the most prominent constituents of the seaweed belt on rocky shores, brown algae of the orders Fucales and Laminariales dominate the intertidal and the subtidal regions, respectively (76).
1.2.1. The order Laminariales
To date, the order Laminariales comprises 142 species (42) of large-sized brown seaweeds, commonly known as kelps. Kelps can reach up to 60 m in length and display a strong morphological thallus differentiation into holdfasts, stipes, and blades, which has been shown to translate into biochemical gradients (76–80). Though not considered taxonomically diverse, kelps are highly distinct structurally and functionally (44), and the only marine algae known to possess specialized cells for the transport of nutrients (81). Kelp species often co-exist within forests, which lie along temperate and polar coastlines, representing some of the most diverse and productive habitats on Earth, providing shelter and serving as food for a great number of associated organisms (44). They are also an economically important resource for humans, with a vast array of applications in different industrial branches, such as food, textiles, and pharmaceuticals. Owing to their rich
polysaccharide composition and the current demand for clean, non-fossil fuel-based energy production, kelps have been thrown into the limelight as potential sources of biofuels (bioethanol) (45,82).
Within kelp forests, the members of the family Laminariaceae (e.g., Laminaria
hyperborea (Gunnerus) Foslie and Laminaria ochroleuca Bachelot de la Pylaie), are
generally the dominant canopy formers of the North East (NE) Atlantic subtidal rocky reefs (83). In the coastal waters of Portugal, kelps are only found associated with areas of intense upwelling, in which Saccorhiza polyschides (Lightfoot) Batters, L. hyperborea, L.
ochroleuca and, sporadically, Saccharina latissima (Linnaeus) C.E. Lane, C. Mayes,
Drueh, are the most important ones (84).
L. ochroleuca is a warm-temperate kelp species, morphologically characterized by a
large heavy holdfast that gives rise to a rigid stipe, which tapers as it approaches the blade (Figure 2A) (85). The genus Laminaria J.V. Lamouroux was taxonomically re-organized by Lane et al. (81), reinstating the genus Saccharina Stackhouse to embrace 18 species formerly included in Laminaria. As with most kelps, S. latissima (formerly Laminaria
saccharina (Linnaeus) J.V. Lamouroux) has a strong morphological differentiation,
exhibiting a long undivided frond with a frilly undulating margin (Figure 2B) (45). This kelp is biogeographically widespread and usually found from the sublittoral fringe down to a depth of 30 m (85,86). Often found at the margins of dense Laminaria forests, S. polyschides can also be dominant canopy-forming macroalgae along large stretches of the NE Atlantic coastline (87). Although S. polyschides is not a true kelp of the order Laminariales, but rather a pseudo-kelp of the order Tilopteridales, it serves a similar ecological function, being commonly treated as a kelp (45). Contrary to L. ochroleuca and S. latissima, two perennial kelp species, S. polyschides is an annual species, also displaying distinctive morphological features (a large warty holdfast, a flattened stipe with a frilly margin and a large blade divided into ribbon-like sections) (Figure 2C) (85).
INTRODUCTION
12
Figure 2. Laminaria ochroleuca (A), Saccharina latissima (B), and a young specimen of Saccorhiza polyschides attached to rocks (C). Photographs provided by Pereira, L. (2018). MACOI – Portuguese Seaweeds Website (MARE, University of Coimbra), available online at http://macoi. ci.uc.pt/.
From a human health perspective, extracts of L. ochroleuca have been found to act as
a central nervous system (CNS) depressants with slight analgesic activity (88), also displaying anti-inflammatory effects (89). S. latissima extracts have also demonstrated several promising bioactivities, including anti-hyperglycemic (90), anti-inflammatory (91), anti-obesity (91), anticoagulant (92), antimicrobial (93), and antioxidant (94,95). Extracts of S. polyschides have shown antiprotozoal (96), antioxidant (58), anti-hyperglycemic (58), and more recently, anti-obesity (97) potential.
1.2.2. The order Fucales
Brown macroalgae of the order Fucales thrive across the rocky intertidal shores all around the world, particularly in cold-temperate regions, producing almost monospecific belts (4). Although to a different extent than Laminariales, brown algae of the order Fucales are also key structuring species, adjusting physical and biological factors within the colonized habitat and promoting biological diversity (98). Besides the ecological roles, Fucales is also pointed out as an economically promising group, with a vast array of applications (fertilizers, food products, drugs, cosmetics).
In the cold-temperate waters of the Northern hemisphere, the genus Fucus Linnaeus is widely spread, and it is, undoubtedly, the most prominent and species-rich genus within the order Fucales. It currently comprises 71 taxonomically accepted species (42), among which Fucus vesiculosus Linnaeus (Figure 3) is, by far, the most well-known and the lectotype species (42).
Morphologically, thallus differentiation of Fucales is generally less pronounced than that of Laminariales, most sections of the thalli being photosynthetically active. In Fucus species, however, a clear distinction into holdfast, stipe, and blade also occurs, and intra-thallus variation in photosynthetic activity and pigmentation has been documented (76). The overall structure of Fucus spp. consists of a flattened, dichotomously-branched thallus that has a small stipe and a holdfast. The blades usually exhibit a central-thickened area (the midrib), and air-filled bladders can be found next to the midrib to keep the seaweed floating upright in its rocky anchorages (Figure 3), as it happens in F. vesiculosus (85).
Although the taxonomy of the Fucus genus is fairly established, new species are occasionally described; this is the case of Fucus guiryi Zardi, Nicastro, E.S. Serrão & G.A. Pearson that was elevated from variety (Fucus spiralis var. platycarpus (Thuret) Batters) to species level few years ago (99).
Figure 3. Fucus vesiculosus (A) and its air-filled bladders (B). Photographs of Mariana Barbosa.
Fucus is one of the oldest genera of macroalgae described and its uses are vast: species
of Fucus are edible and their extracts have been incorporated into cosmetic preparations, and used in seaweed baths, for hundreds of years (85,100). Fucus spp. have also been described as an alternative to chemical pesticides for the management of plant diseases (101,102), but their most popular application, particularly of F. vesiculosus, one of the strongest iodine accumulators of this algae genus, is in the treatment of underactive thyroid glands (hypothyroidism) and goiter, a swelling of the thyroid gland caused by
INTRODUCTION
14
iodine deficiency (103). Several other bioactivities have been ascribed to Fucus spp. extracts, including their well-recognized antioxidant effects (58,104–106), as well as anti-hyperglycemic (58,90), anti-inflammatory (107,108), anti-cholinesterase (58), antiprotozoal (96), anti-hypertensive (66), and anti-tumor (109–111) potential.
1.2.3. Functional compounds
The discovery and development of marine bioactive compounds is a relatively new area when compared to the discovery of those from terrestrial sources. However, research in marine natural products of seaweeds has experienced significant advances in recent years, and screening of multimodal acting compounds to improve human health is indeed at the forefront of scientific innovation. In general, bioactive compounds obtained from seaweeds are structurally and chemically diverse, making their isolation, purification, and subsequent biological testing of single bioactive compounds often difficult (108). Obtaining pure compounds is an expensive process, and isolated compounds rarely have the same degree of activity as an extract, at comparable concentrations or dose of the isolated single constituents (112). The superior effectiveness of many herbal drug extracts used in traditional medicine, in comparison to the single components thereof, can be the result of synergism phenomena between the overall constituents of the extract (113). Besides, a shift in the “one drug, one target” paradigm to multitarget approaches has been encouraged as potential strategies for the management of diseases of multifactorial etiology and complex pathophysiology (114). The multitarget capacity of natural compounds can be, therefore, a promising therapeutic asset.
The employment of new, faster, and environmental-friendly technologies is becoming a primary concern in laboratories devoted to the extraction of natural compounds; however, when working with marine organisms, the conventional solvent- and time-consuming extraction techniques are still the most common practice around the world. In general, it is more challenging to obtain large quantities of bioactive compounds from marine organisms than from terrestrial species. It is therefore understandable that exhaustive extraction procedures, like Soxhlet extraction and maceration, are still used to extract marine compounds (115). Extraction from the algal biomass is also technically challenging and is usually not selective, resulting in complex mixtures of major compounds, namely fatty acids, photosynthetic pigments, phenolic compounds (i.e., phlorotannins), among others (116).
